Viruses claimed a significant component of the nearly 15 million lives lost to infectious diseases in 2002. Viral transmission can occur through several routes, including contact with infected individuals, ingestion of contaminated food and water, or contact with a vector like mosquitoes and ticks. These pathways, which may only need to transmit a few tens to hundreds of viruses to trigger an infection, will only become more prevalent as the globalization and urbanization of our planet accelerates. Thus, the ability to detect these and many other pathogens and disease markers rapidly and at very low levels stands as an extremely challenging proposition central to pubic health monitoring, food/water safety, and bioterrorism. While recent breakthroughs have led to the capability to detect viruses and other nanometric targets (e.g., proteins) at single and double digit levels after capture on a solid phase, the time required for sample/label incubation remains a bottleneck for transitioning to the surveillance/monitoring arena. This proposal seeks to redefine assay speed by exploring the potential of nanoporous gold (NPG) to function as a flow-through capture substrate for the efficient extraction of viruses and other comparably-sized pathogens and disease markers (e.g., antibodies), while at the same time accounting for other considerations needed for effective performance. The basis for this strategy rests with predicted improvements in the mass transfer rates, and thus the binding rates, for both the capture and labeling steps in heterogeneous assays that may be realized by flow through a nanoporous material. Models project potential increases in binding rates of more than two orders of magnitude with respect to the most effective of the known approaches. Two groups of experiments are therefore planned to assess this possibility using gold nanoparticle-based surface enhanced Raman scattering (SERS) measurements. In the first group of experiments, NPG membranes of varied pore size will be fabricated, derivatized, and tested as flow through extraction phases for the model virus feline calicivirus, FCV. FCV, which has ~30-nm diameter and is an effective norovirus simulant, will enable an in-depth, systematic assessment of extraction with respect to pore size. These studies will also test the effect of flow rate on capture and label efficiency, and collectively will provide a set of predictive rules for performance optimization in other potential applications. In addition, experiments will be conducted to minimize the impact of nonspecific adsorption by use of blocking agents, as well as potential complications from membrane clogging through the incorporation of sample prefilters. In the second group of experiments, these guidelines will be applied to assays for the detection of FCV in several matrices, including whole goat serum, tap water and groundwater. PUBLIC HEALTH RELEVANCE: This grant proposal seeks to redefine the speed of heterogeneous immunoassays by exploring the potential of nanoporous gold (NPG) membranes to function as flow through capture substrates for the rapid, efficient and selective concentration of nanometrically-sized pathogens (e.g., viruses and proteins). The basis for this strategy rests with: (1) the predicted improvements in the mass transfer rates, and thus the binding rates, for both the capture and labeling steps that may be realized by flow through a nanoporous material;and (2) the high sensitivity of a readout technique that uses modified gold nanoparticles and surface enhanced Raman scattering (SERS). To carry out the above tasks, we have assembled a team of scientists and engineers from the University of Utah Departments of Chemistry, Chemical Engineering, and Bioengineering, and from the Arizona State University School of Materials.